Composite sensor assemblies for single use bioreactors

ABSTRACT

A composite sensor assembly for monitoring bio-processes which is suitable for use with a polymeric bioprocess vessel or with downstream equipment, and comprises:
         i) a port comprising a high surface tension thermoplastic having a hollow tubular portion and a base plate portion, the base plate being fusibly sealable to the bioreactor vessel at a hole in the wall thereof;   ii) a generally opaque polymeric monitoring sensor assembly including electrical, and/or optical measurement components. The sensor assembly fits inside the bore of the hollow tubular portion of the port, and is adhesively retained therein.

RELATED APPLICATIONS

This application claims priority under 35USC 119(e) from co-pending,commonly assigned Provisional Application Ser. No. 61/465,849 filed Mar.25, 2011 the entire disclosure of which is incorporated herein by thisreference.

BACKGROUND OF THE INVENTION

The increasing popularity of single-use systems for bio-processing isapparent in the market place and can be readily understood byconsidering a typical biotech manufacturing facility. The infrastructurerequired to implement a facility using traditional glass/steelbioreactors, mixers, and purification systems is substantial, and thetime and expense required to construct such bio -process systems can beimmense. The requirement that both the equipment itself and also theingress and egress tubing utilize inert materials such as 316Lelectro-polished stainless steel requires a large initial investment andthe bioreactors, mixers (i.e. bio-process vessels) and down-streamprocessing equipment all have a considerable footprint. In contrast, thesize and form factor of single-use platforms generally permits easierstorage and also re-configurability when compared to traditional, rigidglass/steel solutions. Other advantages of single use systems include alower support infrastructure and time savings over traditional designsincluding specifically the reduction in preparation and sterilizationtime, the reduced need for purified water for cleaning the vessel aftera run, and the significantly reduced post run maintenance time.Additionally, single use systems and their associated plastic tubinglend themselves to being re-configured and validated quickly andefficiently as manufacturing or process requirements change. In thecontext of the present invention we will focus primarily on single-usebioreactors, but the principals apply generically to any of theaforementioned single -use equipment used in bioprocessing.

Although a number of different styles of single use bioreactors havebeen conceived and introduced into the marketplace, two types currentlypredominate. The first type of single-use bioreactor to becomecommercially popular is generally referred to as the “pillow” or“rocker” bag style, and is described, for example, in U.S. Pat. No.6,190,913 the teaching of which is incorporated herein by thisreference. This style of bag can be constructed from a variety ofdifferent polymeric materials, but generally speaking, low or ultra lowdensity polyethylene materials for at least the innermost layer of thebag, i.e., the bag surface which is in contact with the aqueous growthmedium. Other materials sometimes used in the construction of the single-use bioreactor vessels include high density polyethylene (HDPE) andKevlar (Poly -paraphenylene terephthalamide). The pillow or rocker typeof single-use bioreactor utilizes a wave motion induced by movement of abag support platform which generally rocks about a single axis to bothmix and sparge (aerate) the contents of the bioreactor. While the rockertype single-use bioreactor bag has enjoyed considerable marketplacesuccess, to date one major issue has been the lack of robust, single-usesensors that can be integrated into these rocker bags and preferably beradiation sterilized together with the bag. By robust, we mean accurate,gamma or beta radiation stable and capable of being used for real time(real time within the speeds or time responses required forbio-processing) e.g.: 1-3 second sampling process monitoring and controlfor at least 21 days. The pillow or rocker bag is not the only type ofsingle-use bioreactor vessel in use today. A second type imitates theestablished stirred tank reactor. There are single-use polymeric hardshell bioreactors that functionally imitate small scale glass vessels,and also larger scale single-use, plastic liner bags that fit insiderigid pilot and production scale glass/stainless steel stirred tankbioreactors (see e.g., U.S. Pat. No. 7,384,783 the teaching of which isincorporated herein by this reference). The larger liner bags aretypically constructed of films _(I)and laminates that also utilize ultralow density polyethylene or EVA for at least their inner layer. FIG. 6shows the construction of the CX-14 film used by Thermo FisherScientific. Sensors are generally introduced into these largersingle-use bioreactors through lateral ports. Both pillow (rocker) bagsand liner bags can be considered to be “polymeric bioreactor vessels”for purposes of the utilization of the bioprocess monitoring assembly ofthe present invention.

One key issue affecting polymeric bioreactor vessels in general has beenthe method by which to introduce sensors and ancillary monitoringequipment or assemblies that require multiple different materials. (i.ee.g.: mechanical assemblies). The sensors (both single-use andtraditional) are often introduced through the type of prior art portsshown in FIG. 1 (see e.g. published application US2006/0240546) or as isshown in FIG. 2 specifically for a rocker bag. The port shown in FIG. 1can be used to introduce into the vessel a monitoring assembly whichallows the use of different types of sensing elements while the portshown in FIG. 2 is generally restricted to fiber optic based, single-usesensor systems. The port shown in FIG. 1 is typically made of a materialthat is similar to the surface of the bag that it is in contact with, asthis allows it to be readily fused (e.g., thermally or ultrasonicallywelded) to the bag surface. The port shown in FIG. 1 is comprised of acylindrical tube 10 and a flange 11. This type of port uses a mechanicalseal to prevent leakage around the generally cylindrical sensor or otherobject introduced into the tube portion of the port. This mechanicalseal can be a friction fit created by surface to surface contact over arelatively large area as shown in FIG. 3 (see published application US2006/0240546). FIG. 3 shows in more detail how a cylindrical object(e.g., a conventional 12 mm diameter electrochemical dissolved O₂ (DO)or pH probe or aseptic connector like a KleenPak™) 34 fits into thetubular member 31 with a large contact area 33. This prior art port hasa feature 32 that emulates an O-ring and an annular flange 35 which iswelded to the liner of the single use bioreactor vessel. Similarly, theport can actually utilize an O ring seal as shown in FIG. 4, which showsa single use, free space optical assembly (e.g., single use sensorsheath) 41 installed in tubular port member 42 with a weldable polymerflange 43. The O-rings 44 are shown as residing in grooves 45 in theport tubular member. While these port designs can generally provide anair and water tight seal between the port and introduced assemblies, asignificant amount of time is required to qualify and test theseassemblies (e.g.: validate for cGMP use) and they cannot be simply anddirectly assembled when manufacturing single-use polymeric bioreactorvessels. Additionally, there are circumstances where it is difficult todesign a suitable port assembly to support the sensor assembly. This isespecially true in the case of rocker type single-use bioreactor bagswhere there are drawbacks to using fiber optic based single-use sensors,but introducing an optimally designed free space based optical sensorassembly as described in U.S. Pat. No. 7,489,402, the teaching of whichis incorporated herein by this reference, might require a larger port.However, a large port can put stress on the bag materials and istherefore difficult to construct such that the integrity of the bag canbe assured while at the same time maintaining a leak free seal.

A more general method of introducing sensor assemblies, or other type ofmonitoring assembly, into single-use bioreactor vessels would be tosimply weld these assemblies directly into the bags in a manner similarto the way that prior art ports and vents are presently attached tosingle-use bag liners. To date, this has not generally been feasible formost sensors or sensor assemblies. The reasons for the inability toimplement such a straightforward solution for introducing sensors andother accessories into single-use bioreactor vessels include the factthat the bioreactor bags or liners are generally made from laminatedfilms, where the inner layer is typically a high surface tension polymersuch as ultra low density LDPE or EVA; the material used for the sensor(e.g.: whether a free space optical sensor, or electrical sensor)assemblies is generally a polymer such as a polycarbonate, cyclo-olefin,copolyester, or other thermo plastic that is either transparent oropaque, substantially rigid, can meet USP Class VI standards, and inparticular, can withstand the 50 kGy of gamma or beta radiation as isnormally used for sterilization without a significant change in itsphysical or optical properties (e.g., the materials cannot becomebrittle or change opacity). The laminated films used to make the bags orvessel liners can be readily welded together, and although the prior artports which are typically constructed from materials matching the innerlayer of the single-use bioreactor (e.g.: LDPE, EVA, PVDF, or otherpolyolefin) can also be welded to the bag or liner, the optimal materialfor the sensor assembly itself cannot be readily welded to the filmliner materials or to a port of the same material as the liner (See:Materials of Construction for Single-Use Bioprocessing Systems, WilliamHartzel, Innovations in Pharmaceutical Technology, p46, April 2007). Wehave found that the two materials cannot be melted or glued togetherwithout altering the surface at least of the contact layer of the linerand generally without altering both material surfaces. Therefore, theability, as is enabled by the current invention, to construct complexassemblies that can be welded directly to the bag provides important newpossibilities for putting sensors or assemblies in single-use bioreactorvessels and addresses the many issues present with existing sensor portsolutions.

In addition, in order to fully enable the single-use paradigm andprocess optimization on a global scale, the automation software,hardware, and single-use sensors must be expanded from upstreamprocessing (USP) units such as mixers and bioreactors to downstreambioprocessing (DSP) tools such as chromatography assemblies andfiltration skids which utilize similar films. The advent of flexible,modular equipment with integrated data historization would allow thecollection of a unified set of process data from buffer mixing to theultra-filtration. The availability of data from sensors of specificprocess modules (e.g., mixer, bioreactor, and different processconfigurations, especially on the downstream side, would allow users todevelop models for each process step and the interactions therein. Oncesufficient information becomes available from the database, thebio-process engineer could optimize the entire process end-to-end andimplement yield modeling.

In DSP the equipment would ideally implement single-use sensorsfabricated using the manufacturing processes of the present invention asdescribed herein to either replace traditional sensors and/or enable newadditional analytical capability. DSP equipment that utilizes similarfilm technology is described in U.S. Pat. No. 7,935,253. Ideal “smart”sensors for the DSP as well as the USP would have the capability ofbeing pre-calibrated and gamma or beta irradiated along with thebio-process vessel itself In this way the sensors would arrive in thetransport container together with the bio-process vessel. Thus, theentire system arrives sterile, thereby minimizing operator time duringprocess setup. For example, in downstream applications, there is also asignificant need for measuring pH and temperature, as well as opticaldensity and product purity (e.g., viral load, biological impurities). InDSP sensor design, the ability to combine composite materials is evenmore important as the optical requirements in the ultraviolet rangefurther significantly limit the materials choices, and whererequirements on extractables and leachables are becoming ever morestringent. This ability to utilize material combinations that werepreviously considered incompatible from a bonding perspective is animportant enabling factor in both DSP and USP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 are prior art while FIGS. 8-16 are illustrative of the presentinvention.

FIG. 1 shows a prior art port design.

FIG. 2 shows the typical prior art method of introducing fiber opticbased sensors into a single -use rocker bag.

FIG. 3 shows a prior art port design with a seal based on a frictionfit.

FIG. 4 shows a prior art port design with sealing based on an o-ringcompression seal.

FIG. 5 shows another version of a prior art port that utilizes fiberoptic delivery and collection of the optical signal.

FIG. 6 shows the construction layers of the Thermo Fisher CX-14bioreactor film

FIG. 7 is a diagram showing yet another version of a prior art port thatutilizes fiber optic delivery and collection of the optical signal.

FIG. 8 is a top lateral view of a sensor-unit for rocker bags inaccordance with the present invention that includes both optical andthermal sensor windows.

FIG. 9 is a cross-sectional view of a sensor unit in accordance with thepresent invention

FIG. 10 shows a composite sensor assembly i.e. the sensor unit and theport in accordance with the present invention that is thermally weldedto a single-use bioreactor bag.

FIG. 11 shows a partial interior view of a free space optics sensorassembly in accordance with the present invention that canadvantageously replace current prior art port designs on a single-usestirred tank liner bag.

FIG. 12 is an exterior view of the sensor assembly of FIG. 11.

FIG. 13 is a profile view of an optical sensor assembly in accordancewith the present invention suitable for optical scattering or absorptionmeasurements.

FIG. 14 is a cross sectional views of a composite optical sensorassembly in accordance with the present invention, (in this instance anATR spectroscopic device).

FIG. 15 is a cross sectional view of a composite optical sensor assemblyin accordance with the present invention such as an ISFET where theoptical device is in contact with the contents of the bioreactor vessel.

FIG. 16 is a cross sectional view of another type of composite opticalsensor assembly in accordance with the present invention suitable forutilization of NIR or Raman spectroscopic interrogation of the vessel.

FIG. 17 is a cross-sectional view of drive assembly from U.S. Pat. No.7,384,783. Such a drive assembly can be substantially improved iffabricated from a combination of materials which can only be bondedutilizing the process of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The fiber optic based fluorescence sensors currently used in single-usebioreactors are known to suffer from a number of limitations includingthe following:

-   -   1. Accelerated photo-degradation leading to limited lifetime        and/or measuring accuracy/capacity;    -   2. Sensitivity to ambient light;    -   3. Sensitivity to movement or physical perturbation.

Stirred tank single-use bioreactor liners currently utilize portsconfigured, for example, as shown in FIGS. 3 and 4 to introduce sensorsand sampling systems. However, these ports limit the form factor andshape of the sensors that can be utilized.

In the specific case of rocker type single-use bags, the fiber opticbased sensors suffer significant limitations due to the method by whichthey are introduced into the bag. As shown in FIG. 2, the DO (dissolvedoxygen) and pH fluorescent sensor spots are attached to the end of fiberoptic assemblies 22 and 23. These fibers are introduced through the wallof the rocker bag 21, typically near the axis of rotation 24. As shownin FIG. 2, the fiber optic cables are generally brought in through thetop of the rocker bag and the sensor at the end of the fiber optic cableis immersed in the bioprocess media at the bottom of the bag, in afashion similar to a clunk line in a fuel tank. As the vessel rocks backand forth to the maximum angle, the fluid 25 moves to one or the otherend of the vessel and can thus cause the sensor spots to becomeuncovered. As can be seen, this arrangement can cause the sensor spot tobe exposed to the headspace gas (as opposed to being continuouslyimmersed in the aqueous bioprocess media) during a rocking cycle, aswell as being exposed to ambient light which can accelerate thephoto-degradation of the sensor. This is because rocker vessels aregenerally filled to less than half of their capacity so that the liquidcollects in the corners of the bag during the transition end points ofthe rocking cycle leaving the optical sensors situated near the middleof the bag uncovered for a fraction of each rocking period.

Sensor “spots” are typically comprised of fluorescent dyes (see J.Lakowicz, Principles of Fluorescence Spectroscopy, 3^(rd) Edition,Springer, 2006) impregnated in inert, porous materials like cellulose orormosil glass, and the target analyte in question diffuses into thesensor spot and changes the fluorescent properties of the dye(s). Thediffusivity of a gas into the sensor spot is far higher than that of aliquid, so it is easy for even a brief (temporal) exposure to theheadspace gas to affect the sensor reading and thereby cause significantmeasurement inaccuracies. For example, if the response time (90%response) of the sensor spot in liquid is 30 seconds and the DOconcentration in liquid is 30% Sat (saturated), and the response in gasis 3 seconds and the DO is >100% Sat it is easy to see how even briefexposure to the headspace can have a large adverse effect on theaccuracy of the measured DO value.

Several attempts have previously been made to address the aforementionedissues. One proposed solution (see for example U.S. Pat. No. 7,824,902)to the ambient light issue in single-use bioreactors that utilizes fiberoptic based sensors is shown in FIG. 5. Here 51 is the oxygen sensitivefluorescent dye, 52 is a biocompatible material such as low densitypolyethylene that comprises a port which is thermally or RF welded intothe bioreactor's lining 56. The fiber optic cable is shown as 54 while55 is a shield that seeks to prevent ambient light from directlyimpinging upon the photosensitive dye while still allowing fluid to flowfreely around the sensor spot. The fiber, 54, is shown as locking to aferrule, 57, which is part of the port assembly, while 58 indicatesopenings into a receiving vessel which sits above the bag liner. Thissolution allows for affixing the fiber optic assembly and sensor spot tothe single-use bioreactor and also allows for a light shield to limitthe ambient light that impinges upon the spot. This would be of specificimportance to sensor spots like pH spots which typically cannot supportan opaque coating since there are few, if any, opaque, USP Class VIqualified coatings that allow ions (e.g., H⁺) to pass through to thesensor spots.

The rocker type bags and the liner type single-use vessels are generallyconstructed from laminated films as shown in FIG. 6. This figure showsthe Thermo Fisher CX-14 film where the layer in contact with the processis A1 (low density polyethylene, 10.4 mil thick) followed by layer A2 (a0.9 mil thick “tie layer” which bonds A1 and A3), and layer A3(Ethylene-vinyl alcohol copolymer “EVOH”, 1.0 mil thick), and layer A4(another 0.9 mil thick “tie layer” which bonds A3 and A5), and finallyA5 (polyester, 0.8 mil thick). Reference: Thermo Scientific Hyclone BPCProducts and Capabilities 2008/2009. Not all laminated films employed insingle -use applications need to employ these materials or this numberof layers.

Other prior art attempts have been made using a similar method tominimize the time that the sensor is uncovered in a single-use rockerbag by bringing the fiber optic sensors in through a port in the bottomof the bag as shown in FIG. 7 (taken from: Bioprocess Bags IntegrallyEquipped with Oxygen or pH Sensors, Mark Timmins, Si Chen, Aaron Loren,Steven Archibald, Kurt Christoffersen, Jean-Francois Hamel, and JamesKane). However, in this approach the sensor spots can still be exposedto both headspace gas and to ambient light thereby allowing themeasurement fidelity to be compromised and the sensor spot to be subjectto photo-degradation. The spots are at, or slightly above the bottom ofthe bag. The fiber optic cable, 64, is connected to a standard fiberoptic connector, 63, which is embedded in a port 62 which is thermallywelded to the inner film layer (normally LDPE or EVA or similar inertlayer) of the single-use bioreactor 61. The sensor spot, 60, is attachedto the end of the fiber optic cable. However, we have found that withthis design for low liquid volumes the spots can still become uncoveredat the maximum rocking angle and speed. Also, in this approach there isno protection for the sensor spots from the effects of ambient light.

An optimal system would avoid the known issues of fiber optic cablebased optical sensors: it would minimize ambient light exposure, and itwould also eliminate the possibility of having the values measured bythe sensor when immersed in liquid being corrupted by exposure toheadspace gas phase contributions. Ideally, the system would also becapable of being attached to the bag during the normal course ofconstruction using the same equipment (e.g.: thermal welding equipment,seam sealers) used to make the bag. The present invention addresses andsolves these issues.

We have found that illumination based photo-degradation issues can beaddressed by using a free space optics based sensor design. The use offree space optics and the details of such sensors and their advantagesare described in U.S. Pat. No. 7,489,402 the entire teaching of which isincorporated herein by this reference. A fundamental concept put forthin this patent, is that the drift in measurement accuracy in phasefluorimetric sensor systems due to photo-degradation can besignificantly reduced (in many cases to a level that is not measurable)by minimizing the amount of light used to illuminate the sensor spot(s).Free space optics and an appropriately sized photo-detector can, invirtually all circumstances, collect far more light from a fluorescentsensor spot than can a fiber optic cable. As is known, fiber opticsystems are limited by fundamental physical laws as to how much lightthey can collect. The fundamental law of physics describing this issueis known as “conservation of brightness”. This limitation also appliesto free space optics, but the limitation's effects are nowhere near assevere. As a free space optical system can collect far more light(easily greater than a factor of 10 vs. a fiber optic cable) it can usefar less light to illuminate the fluorescent sensor and will thereforecause a far slower rate of photo-degradation. However, for optimallyaccurate results a method is needed to minimize or eliminate theexposure of the spot to the gas phase and also ensure that the entirespot always remain submerged, even for low fill volumes and to alsoprotect the spot from exposure to ambient light.

A sensor design in accordance with the present invention, which isparticularly suitable for use in rocker bags, and which can bephysically realized and practically implemented to avoid theaforementioned problems, is shown in FIGS. 8 through 10.

The composite sensor assembly of the present invention is suitable foruse with a polymeric bioprocess vessel or with downstream bioprocessingequipment, and comprises:

-   -   i) a port comprising a high surface tension thermoplastic such        as LDPE or EVA having a hollow tubular portion and a base plate        portion, the base plate being fusibly sealable to the bioreactor        vessel at a hole in the wall thereof;    -   ii) a polymeric sensor (monitoring) unit including electrical        and/or optical measurement components (generally opaque when        only optics are used). The sensor unit fits inside the bore of        the hollow tubular portion of the port, and is adhesively        retained therein using a special process as described        hereinafter.

The port provides the sensor unit components with access to the contentsof the bioreactor vessel, which components include means for providingincoming optical and/or electrical signals and means for collecting andtransmitting measurement signals emitted by the sensor assemblycomponents. For use with a rocker bag, the sensor unit is preferably(but not necessarily) circular and is generally dish shaped and includesa base portion and a concentric rim extending above the base. The sensorunit has cup shaped depressions in the inner surface of the basethereof. Each of the monitoring components is situated in a separate cupshaped depression in the base of said sensor unit and each said cupshaped depression preferably includes a surrounding rim which extendsabove the inner surface of the sensor unit base. The cups containingoptical sensor (monitoring) components will preferably have a lightshield positioned over the top thereof. In current practice, althoughnot required, particularly in a free space optics sensor unit designedfor use with a liner bag the probe will generally be substantiallycylindrical in shape and be contained within the bore of the tubularportion of the port as shown in FIGS. 11 and 12 the sensor unit againbeing adhesively retained therein using a special process as describedhereinafter.

FIG. 8 shows the free space optical sensor unit in which the DO and pHspots are mounted in the recessed “cups” 72 and 73 which help maintainbioprocess fluid covering the spots at all normal angles of rocking androcking speeds. The temperature is measured through a cup having a thin316L electro-polished stainless steel plate shown as 71.

FIG. 9 is a cross-sectional view of a sensor unit in accordance with thepresent invention for use with a rocker bag showing the optically opaquesections of the sensor unit 82, as well as the optically transparentsections 83. The opacity is normally achieved by incorporating USP ClassVI absorptive colorant into a normally transparent or translucentpolymer. It should be noted that the entire sensor unit in certainembodiments will preferably have a peripheral ridge 84 which serves toretain fluid inside the sensor unit. An additional benefit of the ridgeis that it creates a fluid flow path which minimizes the collection ofparticulates (e.g.: dead cells or other precipitates) in the sensorcontaining cups 85. As shown, each sensor sits below the fluid surfacein its own cup 85. The temperature sensor containing cup 87 is normallyslightly different in size and depth compared to the pH or DO sensorcontaining cups, but follows the same concept in that it has beendesigned to retain liquid. Instead of a free space optical sensor itutilizes a 316L electropolished plate for good thermal conductivity withthe bioreactor fluid and contains an RTD, thermistor or equivalenttemperature sensor. Note that each of the cups 85 also preferably hasits own ridge 86 which similarly alters the liquid flow through of thecup and thereby reduces the collection of debris in the cup. The ridgescreate a flow pattern conducive to sweeping out debris.

The materials chosen for the polymeric portions of the sensor unit,including the cups that hold the sensor spots will advantageously havethe following characteristics:

-   -   Stability (in optical and mechanical properties) after exposure        to sterilizing gamma or beta radiation;    -   Structural integrity;    -   Low cost;    -   Ability to meet USP Class VI standards;    -   Animal component derived free;    -   Ability to be molded;    -   Optical opacity of the body of the sensor unit and optical        transparency in the portions that need to transmit light.

The list of polymeric materials that meet the above criteria includespolycarbonates, cyclo-olefin copolymers, co-polyester, polystyrene andother beta or gamma radiation stable thermo-plastics.

As previously mentioned the innermost layer of single-use rocker bags isgenerally constructed from LDPE or EVA because these polymers arechemically inert and non-reactive with biological materials and also areavailable as a USP Class VI film. Additional outer reinforcing and/orbonding layers are sometimes also present so that many bags includelayers of other polymers in addition to the innermost LDPE or EVApolymer layer. Henceforth, we will generally refer to LDPE as thematerial used to construct the single-use bioreactor bag but the bag canbe fabricated from one of the aforementioned materials or other suitablefilm meeting USP Class VI standards.

A central issue and problems that are addressed by the current inventionis that most of the materials suitable for the sensor unit cannot bethermally or otherwise readily and effectively bonded to LDPE withoutsubjecting at least the LDPE to surface preparation, and in many casessubjecting both the LDPE and the sensor unit to surface preparation. Itis difficult to bond LDPE to dissimilar materials in a reliable, robustway that still allows the bonded part to withstand sterilization(typically by gamma or beta radiation) and also still meet USP Class VIstandards. This makes it difficult to utilize the optimal materials froma free space optical device (or electrical device or device usingoptics, electronics, and/or chemical transducers) perspective for thesensor unit, while at the same time maintaining the ability to bond thispart (the sensor unit) into the single-use bag. Therefore, it is notobvious how to simultaneously implement a sensor unit that meets therequirements for measurement (e.g.: lifetime, accuracy, functionality)and also sealingly integrate it into a single-use bioreactor vessel.

In order to create an optimal free space optical system for phasefluorimetry, materials meeting the aforementioned requirements must beused. Therefore, a bonding method that meets all the applicablerequirements must be employed to bond the sensor assembly to thesingle-use bioreactor vessel. A variety of methods of surfacepreparation are known and some could in theory be employed with LDPE orEVA (see e.g., “Adhesion enhancement of polymer surfaces by atmosphericplasma treatment”, M J Shenton, M C Lowell-Hoare, and G C Stevens,Journal of Physics D: Applied Physics, 34 (2001) 2754-2760). Forexample, it is possible to chemically etch the surfaces to be mated oralternatively to create a “meta-layer” where the surfaces are doped withmaterials that bond together more readily. While such methods enable thebag and the interfacing surface portion of a sensor assembly to bebonded together, there are very few choices of chemical etchants thatwill provide a product that meets USP Class VI standards. We have foundthat a more readily available method that works for LDPE, EVA and mostother poly-olefins or ethylene copolymers used for bioreactor bags issurface preparation of the mating surfaces of both the portion of thesensor assembly (which is fabricated from a polymer that is readilyfusibly bonded to the bag) and the rigid sensor unit structurecontaining the free space optics, electronics, or a combination thereofby methods such as plasma cleaning, UV ozone cleaning or other approachthat creates adsorption sites on the mating surfaces. As most single-use bags are manufactured by “welding” the parts together (usingthermal methods, ultrasound, etc.), it is therefore advantageous tocreate a part (i.e., the sensor assembly of the present invention) thatcan be welded into the vessel in the same fashion as conventional portsand vents.

Our invention therefore utilizes a port fabricated at least in part fromLDPE, EVA or similar polymer suitable for bonding by known methods tothe single-use liner or rocker bag film (i.e., be readily bondable tothe LDPE bioreactor bag inner layer surface) combined with a free spaceoptical sensor unit fabricated from one of the aforementioned specialpolymers (e.g., polycarbonates, cyclo-olefin copolymers, copolyesters,and polystyrene). The port to bag and port to sensor unit whenfabricated in accordance with the present invention provides an asepticand fluid impervious seal. Following the aforementioned surfacetreatment of either or preferably both of the mating surfaces of theport and the sensor unit, we have found they may be advantageously andeffectively bonded together using USP Class VI adhesives which includeone or two part epoxy resins, UV curable epoxies, cyanoacrylates,silicones, or polyurethanes. In order to ensure that the chosenadhesives do not excessively cross-link and become brittle under gammaor beta sterilizing radiation, they can be first tested after radiationexposure in order to qualify them for this application in accordancewith applicable USP standards.

The composite sensor assembly we describe herein uses the aforementionedsurface preparation techniques to enable the bonding of anoptical/physical component to an LDPE or EVA port which port can besubsequently welded using known methods into a single-use vesselfabricated from LDPE (or EVA). This method enables a far greater degreeof freedom in the design of components that can be sealed intosingle-use bioreactors which utilize materials such as LDPE or EVAhaving a high surface tension that are therefore extremely inert andalso difficult to bond to. This alleviates the need to insert thecomponents through rigid or semi-flexible ports that are limited inshape and spatial extent. The part shown in FIGS. 8 and 9 is a sensorunit in accordance with the present invention that allows for the use offluorescent based optical sensor(s) and also, if desired, a temperaturesensor (thermal probe). Moreover, this method can be applied to otherdifferent types of probes (monitoring devices) that are advantageouslyused with a single-use bioreactor including electrical sensors (e.g.:ISFETS) or other types of optical sensors based on spectroscopic methods(e.g.: near infra-red or Raman). FIG. 10 shows the composite sensorassembly in accordance with the present invention, i.e., the sensor unitintegrated with the port and fused to the bioreactor vessel wall.Additionally, this bonding method is not limited to a rocker typesingle-use bag or polymeric liner bags as the composite assembly of thepresent invention can be readily welded to any single-use polymer vesselthat requires sensors. Finally, this method does not compromise the USPClass VI classification of the materials, so that the combined productis still perfectly suitable for bio-processing applications.

As shown in FIG. 10, the sensor assembly which includes the sensor unithaving optical sensor cups (shown as 72 and 73 in FIG. 8 and as 83 inFIG. 9) and a plate 91 with high thermal conductivity (e.g.: 316Lelectro-polished stainless steel), and which also meets USP Class VIstandards and through which temperature can therefore be sensed ormeasured are mounted to the port base plate 92 (preferably fabricatedfrom LDPE) with suitable adhesive subsequent to plasma cleaning, and theassembly is then thermally welded to the bag film 93 (shown here incircular form). Optional light shields, 94 to avoid signal interferenceand photo-bleaching are also shown situated above the optical sensorcups. The light shield is also advantageously used on fluorescent sensorspots that cannot support or cannot otherwise be used with an opaquecoating such as black silicone. The light shield allows bioreactor fluidflow to the sensor spot, but blocks a large percentage of the ambientlight. This reduces the potential for photo -degradation of the sensorspot and also for interference with the optics and electricalamplification used in the fluorescent signal detection.

FIG. 11 shows one version of the composite sensor assembly of thepresent invention that would be particularly suitable for use with asingle-use, stirred tank plastic liner bag. The sensor unit shown aspart of the sensor assembly in FIG. 11 is comprised of a preferably atleast partially opaque optical sensor unit that is constructed of asuitable USP Class VI, gamma radiation resistant, substantially rigidand animal component derived free polymeric material as previouslydescribed. Here 102 is a transparent component (e.g.: a lens) made froma similar material but without colorant added. A fluorescent sensor spot(not shown) is suitably placed on top of the optical component. Otheroptical sensor measurement techniques like NIR spectroscopy or Ramanspectroscopy require no sensor spot. A suitable thermally conductivearea (e.g., a 316L electro-polished stainless steel plate fortemperature measurement) is shown as 103. The port 104 is suitably madefrom a material that is readily welded to the single-use bag liner andwill preferably have a circumferential flange 105 to facilitate saidbonding to the bioreactor bag. It is sometimes also desirable to haveadditional features in the sensor unit 101 and/or the port 104 (i.e.,have an increased mating surface area or a ledge) such that when theport's inner surface (106) and the sensor unit's outer surface areprepared via plasma treatment, an adhesive such as platinum curedsilicone 107 will readily adhere to both surfaces. The composite sensorassembly as shown in FIGS. 11 and 12 (an exterior, non-cutaway view ofthe sensor assembly of FIG. 11) utilizes a free space optical sensorunit, but is equally suitable for use with an electrical sensor unit.Although the port is shown as being cylindrical in shape the descriptionthereof as being “tubular” is not to be construed as requiring eitherthe tubular portion of the port or the sensor unit to be circular incross section.

FIG. 13 depicts a profile view of a composite optical assemblyparticularly suitable for optical density measurements (UV, nearinfra-red, or visible) or optical absorption spectroscopy measurements.In FIG. 13, 120 is the liner compatible material (e.g.: LDPE), and 121is the single-use bag film. The optical part of the composite assemblyis comprised of the sidewall, 124, which is adhesively affixed to theliner compatible material 120, whereas 123 is the floor of the opticalassembly and can suitably be of the same material as 124 or notdepending on the optical properties required. Components 122 are opticalprisms that are used to refract or reflect the light beam 125 beamacross an optical gap. The prisms would typically be coated such thatthey reflect the light beam 125, as shown. The light traversing the gapin part determines the optical properties of the components utilized andtherefore the material selection. The exact separation between theprisms (optical gap length) is determined by the absorption andscattering properties of the material that is intended to becharacterized by this system. The processing of the optical signals canbe by a photo-detector or power meter with a filter in front, or it canbe by a spectrometer.

FIG. 14 depicts a cross-sectional view of another composite opticalassembly. In particular here we show an ATR (attenuated totalreflection) spectroscopic device. G. Muller, K. Abraham, and M.Schaldach, “Quantitative ATR spectroscopy: some basic considerations,”Appl. Opt. 20, 1182-1190 (1981). In this cross-sectional view, 131 isthe liner or film of the single-use bioreactor, 130 is the LDPE or othercompatible material, 134 is another, suitable material that isadhesively affixed to the LDPE. Component 136 is typically a highrefractive optical index material (e.g.: Al₂O₃ or YVO₄, or high indexglass, polycarbonate, or similar material). The high index is requiredto allow the frustrated total internal reflection. The incoming light isshown here as 135, and the evanescent sites or “bounce sites” are shownas 136. The exact optical refractive index and the number of bouncesites are determined by the sensitivity required the absorptioncoefficient of the analyte in question at the wavelength used and otherfactors.

FIG. 15 depicts a cross-sectional view of a sensor assembly including anelectrical device, 142, (e.g.: an ISFET) that is in contact with thecontents of the bioreactor to provide a reading where 141 is thesingle-use bioreactor film and 140 is the composite's first bioreactorcompatible material (e.g.: LDPE). The composite's second compatiblematerial 143 is adhesively affixed to 140. The electoral element, 142,can be molded directly into 143, adhesively affixed, or held in using amechanical seal such as a compression fit using an o-ring or similartechnique depending on the device and its particular properties.

FIG. 16 shows a cross-sectional view of another type of composite sensorassembly where 151 is the single-use film, and 150 is the composite'sfirst compatible material such as LDPE. The composite's secondcompatible material is shown as 154 and is adhesively affixed to 150.Item 155 represents an optical unit comprised of a light source and adetection system which may utilize a spectrometer or a set of filters toidentify the spectral components of the returned light. The opticalsource will impinge upon 152 which is a generally tubular and is atleast partially transparent to the light source. The tubular element 152is constructed so that the contents of the bioreactor can becontinuously circulated through it allowing accurate, non-invasive,in-situ examination of the bioreactor contents. Circulation can, ifdesired, be facilitated by a pump, 156. The system shown can be used toperform near-infrared absorption spectroscopic measurements, or Ramanmeasurements of the bioreactor contents.

Earlier the suitability and advantages of fabricating compositemechanical assemblies for polymeric bioreactor vessels utilizing thebonding procedures of the present invention was indicated. This isbecause many of the liner style single-use bioreactors (and mixers) relyon a motor driven agitator to mix and help aerate the contents of thevessel. The agitator shaft must be introduced into the bioreactor vesselthrough a port which also constrains the shaft. A similar fundamentallimitation imposed by material compatibility is encountered here aswell. In FIG. 17 an agitation drive unit as described in of U.S. Pat.No. 7,384,783 (the teaching of which is incorporated herein by thisreference) is shown. In FIG. 17, as per FIG. 6 of the aforementionedPatent, is shown a partial cross-section view of a rotational assembly601, where the rotational assembly includes a bearing assembly 670disposed between a hub 620 and an inner casing 660. Additionally, shownis a lower race bearing or the assembly 670 is in a fixed relation withthe inner casing 660. Hub 620 can rotate relative to the race bearingand may include a guide 624 a for receiving snap rings or retainingrings which can help maintain hub 620 in place. As shown the outercasing 661 must be bonded to the single-use vessel wall 699. Thisrequires that the casing 661 be constructed of a material compatiblewith the bioreactor vessel film, or casing 661 must be over-molded witha film compatible material (i.e., bondable to the film). Requiringmaterial compatibility limits the selection of materials to materialssimilar to LDPE which then limits the ability to select casing materialswith the optimal characteristics for the application (e.g.: tensilestrength, elasticity etc, hardness). Over-molding also limits thematerials that can be used. Although ultra low density polyethylene usedfor the films can be sometimes be effectively over-molded onto morerigid high density polyethylene (HDPE) and thereby allow HDPE to beemployed for casing 661 in some cases. However, even HDPE is generallysub-optimal for this application.

As described here, with the proper surface treatment many more suitablematerials can be used for the mechanical unit (e.g.: the hub 620, and/orthe bearing assembly 670) and be adhesively affixed to the outer casing661. This provides an alternative and preferred path to constructing thedrive assembly in a similar fashion to the sensor unit and port aspreviously described.

Additional mechanisms for bioprocessing applications that can benefitfrom the ability to utilize fundamentally different materials includebut are not limited to spargers (aeration devices) and sampling ports.Single use bioreactors employ complex frits or membranes to provide theporous apertures required to make bubbles of certain sizes for gas masstransfer purposes and their design can be limited by the materialselection and compatibility issue that affects sensors. Ports forautosamplers can also require multiple materials, as the materials thattouch the bioprocess fluids might need to be UV transparent in order tobe sterilized in-situ. This is currently impossible using LDPE andsimilar materials as they absorb strongly in this region.

Various modifications and variations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of theclaims.

1-24. (canceled)
 25. A composite assembly for use with a polymericbioprocess vessel, the composite assembly comprising: a bioprocessvessel including a wall having an opening; a port including a highsurface tension thermoplastic, wherein the port includes a hollowtubular portion and a base plate portion, the base plate includes apolymer fused to the bioprocess vessel at the opening in the wall; and apolymeric unit adhesively retained within the bore of the hollow tubularportion of the port, wherein the port provides the polymeric unit withaccess to contents of the polymeric bioreactor vessel.
 26. The compositesensor assembly of claim 25, wherein the polymeric unit is adhesivelyretained within the bore of the hollow tubular portion of the port by anadhesive between interfacing surfaces of said port and said sensor unit.27. The composite sensor assembly of claim 25, wherein the polymericunit includes a monitoring sensor having electrical and/or opticalmeasurement components.
 28. The composite sensor assembly of claim 27,wherein the monitoring sensor includes a generally planar base portionand a concentric rim extending above the base portion.
 29. The compositesensor assembly of claim 27, wherein said polymeric unit has cup shapeddepressions in an inner surface of the base thereof.
 30. The compositesensor assembly of claim 25, wherein the polymeric unit has a generallycircular shape.
 31. The composite sensor assembly of claim 27, whereineach of the electrical and/or optical measurement components is situatedin a cup shaped depression in the base of the polymeric unit and whereineach cup shaped depression includes a surrounding rim which extendsabove the inner surface of the sensor unit base.
 32. The compositesensor assembly of claim 31, wherein at least one of the cup shapeddepressions has a light shield positioned over the electrical and/oroptical measurement components.
 33. The composite sensor assembly ofclaim 26, wherein the adhesive is a USP Class VI adhesive.
 34. Thecomposite sensor assembly of claim 26, wherein the adhesive is selectedfrom the group consisting of one or two part epoxy resins, UV curedepoxies, cyanoacrylates, platinum cured silicones, and polyurethanes.35. The composite sensor assembly of claim 25, wherein said high surfacetension thermoplastic is low or ultra low density polyethylene orethylene vinyl acetate copolymer.
 36. The composite sensor assembly ofclaim 25, wherein the polymeric unit is tubular.
 37. The compositesensor assembly of claim 25, wherein the polymeric unit comprises anopaque sheath.
 38. The composite sensor assembly of claim 27, whereinthe monitoring sensor includes a fluorescent sensor spot.
 39. Thecomposite sensor assembly of claim 38, wherein the monitoring sensorincludes a light shield.
 40. The composite sensor assembly of claim 27,wherein the monitoring sensor includes a free space optical densitymeasurement unit.
 41. The composite sensor assembly of claim 27, whereinthe monitoring sensor includes an optical spectroscopy measurement unit.42. The composite sensor assembly of claim 27, wherein the monitoringsensor includes an ISFET unit.
 43. A composite sensor assembly forbioproduction, comprising: a polymeric bioprocess vessel including anopening; a flexible port comprising a high surface tensionthermoplastic, wherein the flexible port has a hollow tubular portionand a base plate portion and the base plate is fused to the opening inthe polymeric bioprocess vessel; and a polymeric sensor unit includingmeasurement components, wherein the sensor unit fits inside and isadhered to the bore of the hollow tubular portion of the flexible port,wherein the port provides the measurement components with access tocontents of the polymeric bioprocess vessel.
 44. A composite sensorassembly for bioproduction, comprising: a polymeric bioprocess vesselincluding an opening; a flexible port comprising a high surface tensionthermoplastic, wherein the flexible port has a hollow tubular portionand a base plate portion and the base plate is fused to the opening inthe polymeric bioprocess vessel; and a polymeric sensor unit adhered tothe inside of the bore of the hollow tubular portion of the flexibleport, wherein the port provides a fluorescent dye spot on the polymericsensor unit with access to contents of the polymeric bioprocess vessel.